LOCUS       sequence-435694-        8982 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Mammalian expression of CLIPf-KIF5A(1-914; 
            Delta-505-610)-SNAPf-HisTag-FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8982)
  AUTHORS   Smith ER, Turner ED, Abdelhamid MAS, Craggs TD, Twelvetrees AE
  TITLE     Kinesin-1 is highly flexible and adopts an open conformation in the 
            absence of cargo
  JOURNAL   bioRxiv 2024.12.20.629623
REFERENCE   2  (bases 1 to 8982)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.12.20.629623"
COMMENT     Sequence Label: CLIPf-KIF5A(1-914; Delta-505-610)-SNAPf-HisTag-FLAG
FEATURES             Location/Qualifiers
     source          1..8982
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     464..483
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    768..889
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(805..824)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     859..878
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(938..954)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(938..954)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(951..973)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    962..978
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(986..992,993..1010,1011..1016))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1031..1052
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1169..1186)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1340..1928)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1420..1439)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2099..2890,2891..2959))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2722..2741
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2960..3064)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3139..3158)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3330..3709
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3710..3913
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3864..3884
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3958..3977
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3958..3976
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             7579..7596
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7597..7620
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(7668..7685)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7674..7898
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7944..8372
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8031..8050)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8241..8262
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8381..8401)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        8386..8715
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      8566..8701
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     8628..8647
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             join(8782..8982,1..594)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(8836..8855)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
       61 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
      121 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
      181 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
      241 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
      301 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
      361 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
      421 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
      481 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
      541 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
      601 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
      661 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
      721 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
      781 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
      841 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
      901 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
      961 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     1021 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     1081 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     1141 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     1201 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     1261 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     1321 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     1381 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     1441 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     1501 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     1561 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     1621 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     1681 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     1741 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     1801 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     1861 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     1921 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     1981 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     2041 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     2101 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     2161 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     2221 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     2281 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     2341 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     2401 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     2461 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     2521 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     2581 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     2641 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     2701 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     2761 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     2821 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     2881 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     2941 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     3001 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     3061 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     3121 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     3181 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     3241 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     3301 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
     3361 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     3421 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     3481 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     3541 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     3601 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     3661 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     3721 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     3781 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     3841 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     3901 tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa
     3961 tacgactcac tatagggaga cccaagctgg ctagcgttta aacttaagct tggtaccatg
     4021 gacaaggact gcgagatgaa gcggaccaca ctggatagcc ctctgggcaa gctggaactg
     4081 agcggctgtg aacagggcct gcaccggatc atctttctcg gcaagggaac ctctgccgcc
     4141 gatgctgttg aagtgcctgc tcctgctgct gtgctcggag gacctgaacc tctgattcag
     4201 gccaccgcct ggctgaacgc ctactttcat cagcccgagg ccatcgagga gttccccgtt
     4261 cctgctctgc atcaccccgt gttccagcaa gaaagcttca ccagacaggt gctgtggaag
     4321 ctgctgaagg tggtcaagtt cggcgaagtg atcagcgagt ctcacctggc cgctctcgtg
     4381 ggaaatccag ctgctacagc cgccgtgaac acagccctgg atggaaaccc tgtgcctatt
     4441 ctgatcccct gccacagagt ggtgcagggc gattctgatg tgggccctta tcttggaggc
     4501 ctggccgtga aagaatggct gctggcccac gaaggacaca gactgggaaa acctggcctc
     4561 ggaggatcca tggcggagac taacaacgaa tgcagcatca aggtgctttg ccgatttcgg
     4621 cccctgaacc aggccgagat tctgcggggg gacaagttca tccccatttt ccaaggggac
     4681 gacagcgtca ttattggggg aaagccatat gtctttgacc gcgtcttccc cccaaacacc
     4741 actcaggagc aggtttacca cgcctgtgcc atgcagatcg tcaaagacgt ccttgctggt
     4801 tacaatggca caatcttcgc ttatggacag acatcctcag ggaaaacgca taccatggag
     4861 gggaagctgc acgaccctca gctgatgggc atcattcccc ggatcgctcg agacatcttc
     4921 aaccacatct actccatgga tgagaacctt gaattccaca ttaaggtatc ttacttcgag
     4981 atttacctgg ataagatccg tgaccttttg gatgtgacca agacgaacct gtccgtgcat
     5041 gaggacaaaa accgggtgcc gtttgtcaag ggttgtaccg aacgctttgt gtccagccca
     5101 gaggagattc tggatgtgat cgatgagggg aagtccaacc gtcacgtagc tgtcaccaac
     5161 atgaacgagc acagttctcg gagccacagc atcttcctca tcaacatcaa gcaggagaac
     5221 gtagagaccg agcagaagct cagcgggaag ctgtacctcg tggatctggc cggaagcgag
     5281 aaggtcagca agacaggggc agagggagcc gttctggacg aggcaaagaa tatcaacaag
     5341 tcgctgtcgg ccctggggaa cgtgatctct gcactggcag agggcaccaa aagctacgtg
     5401 ccgtaccgcg acagcaaaat gacgaggatt ctccaggact ctctgggagg gaactgcagg
     5461 actaccatgt tcatctgctg ctcgccgtcc agctacaatg acgcagagac caagtccacg
     5521 ctcatgtttg gacagcgggc gaagaccatc aagaacactg cctcagtgaa tctggagctg
     5581 actgctgagc agtggaagaa gaagtatgag aaggagaagg agaagaccaa ggcccagaag
     5641 gagacaattg cgaagctaga ggctgagctt agccggtggc gcaatggaga gaatgtgcct
     5701 gagactgagc gcctggctgg agaggactca gctctgggag ctgagctctg cgaggagacc
     5761 cctgtgaatg acaactcatc cattgtggta cgcatcgcac ctgaggaaag gcagaaatat
     5821 gaggaagaga tccgccgtct ctacaagcag cttgatgaca aggatgatga gatcaaccag
     5881 cagagccagc tcattgagaa gctgaagcag cagatgctgg accaggaaga gctgctcgtg
     5941 tccactcggg gagacaacga gaaggtccag cgggagctta gccacctgca gtccgagaac
     6001 gatgctgcga aggacgaggt gaaggaagtg ctgcaggccc tagaggagct ggcggtcaac
     6061 tacgaccaga agtcccagga gctccaggtg gagtgtcatc gcaagatgga ggtgaccggt
     6121 agggagctgt catcttgcca actgctcatc tcacagcatg aggccaagat ccgttcactc
     6181 acggagtaca tgcagactgt ggagttgaag aaacggcacc tggaagagtc ctacgactcc
     6241 ctgagcgatg agcttgccag gctccaggcg cacgaaactg tacacgaggt agctctgaaa
     6301 gacaaggagc cagacacaca ggacgcggag gaggtgaaga aggccctgga actacagatg
     6361 gagaatcacc gtgaggccca tcaccggcag ctggcccgcc tccgggatga gattaatgag
     6421 aaacagaaaa ccattgatga gctgaaagac ctgaaccaga agctccagtt agagctggag
     6481 aagcttcagg ccgactatga gaggctgaag aatgaagaga acgagaagag cgccaagctc
     6541 caggagctga catttctgta tgagcgacat gagcagtcca agcaggacct caaggggctg
     6601 gaggagacag ttgcccgtga actccagacc ctccacaacc ttcgcaagct gttcgttcaa
     6661 gacgtcacga ctcgagtcaa gaaaagtgca gaaatggagc ccgaggacag tggggggatt
     6721 cattcccaaa agcagaagat ctcctttctt gagaacaacc tggaacagct tacaaaggtt
     6781 cacaaacagc tggtacgtga caatgcagat ctgcgttgtg agcttcctaa attggaaaaa
     6841 cgacttcggg ctacggctga gagagttaag gccctggagg gtgcactgaa ggaggccaag
     6901 gagggcgcta tgaaggacaa gcgtagatac cagcaggagg tggaccgcat caaagaagcc
     6961 gtgcggtaca agagctccgg caagcggggc catgaattcg ataaggattg tgaaatgaag
     7021 cgcacaaccc tggactcccc actgggcaaa cttgagctgt ctggatgcga gcagggactg
     7081 cacagaatca tattcctcgg aaagggcacc agcgccgctg atgcagtgga agttccagca
     7141 ccagctgcag tgcttggtgg accagagcca cttatgcagg ctacagcttg gctgaatgct
     7201 tatttccacc agccggaagc catcgaagag tttcccgtgc cagcactgca ccatcctgtc
     7261 tttcaacaag agtccttcac acggcaagtc ctgtggaaac tcctgaaagt cgtgaaattt
     7321 ggggaagtga tctcctacag ccacctggct gcactggccg gaaatcctgc agcaactgcc
     7381 gctgtgaaaa ctgccctgtc cggcaatcca gtgccaatcc tgattccttg tcaccgggtc
     7441 gtccagggcg acctggatgt tggaggatat gaaggcggac tggctgtcaa agagtggctc
     7501 cttgctcacg agggccatag actcggaaag cctggacttg gtggaagcgc cggatctgca
     7561 gcaggatctg gcgagtttca ccaccaccat caccacgact acaaggacga cgacgacaaa
     7621 tgagcggccg ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     7681 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     7741 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     7801 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     7861 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     7921 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     7981 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     8041 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     8101 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     8161 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     8221 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     8281 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     8341 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     8401 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     8461 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     8521 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     8581 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     8641 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     8701 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     8761 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     8821 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     8881 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     8941 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc ta
//
